Genetic analysis reveals conspecificity of two nominal species of Anaphes fairyflies (Hymenoptera: Mymaridae), egg parasitoids of Oulema leaf beetle (Coleoptera: Chrysomelidae) pests of cereal crops in Europe and of rice in East Asia

Anaphes (Anaphes) flavipes (Foerster), a fairyfly (Hymenoptera: Mymaridae) native of Europe, is an economically important egg parasitoid for the natural control of Oulema spp. leaf beetle (Coleoptera: Chrysomelidae) pests of cereal crops such as barley, oats, rye, and wheat in Europe, and for the classical biological control of the invasive Oulema melanopus (L.) in North America. A morphologically very similar Anaphes (Anaphes) nipponicus Kuwayama, known from mainland China, Japan, Republic of Korea, Far East of Russia and Taiwan, is an egg parasitoid of Oulema oryzae (Kuwayama), a pest of rice mainly in temperate parts of East Asia. The nuclear 28S-D2 and ITS2 and the mitochondrial COI genes were used as markers to compare specimens of A. (Anaphes) flavipes reared from eggs of an Oulema sp. on barley in Germany with those of A. (Anaphes) nipponicus reared from eggs of O. oryzae on rice in Honshu Island, Japan. Because the resulting sequences are practically identical, within an expected intraspecific genetic variability, conspecificity of these two nominal species has been confirmed, and consequently A. (Anaphes) nipponicus Kuwayama, 1932, syn. n. is synonymized with A. (Anaphes) flavipes (Foerster, 1841). Taxonomic notes and illustrations are provided for the specimens of both sexes of A. (Anaphes) flavipes from Japan to facilitate their recognition.


Introduction
Leaf beetles of the genus Oulema Des Gozis (Coleoptera: Chrysomelidae) are rather common pests, mainly in the Palaearctic region, of cereal crops such as barley, oats, rye, and wheat in Europe as well as rice in East Asia. Oulema melanopus (L.), one of the several species of the genus native to Europe, was unintentionally introduced to North America where it became a serious pest of wheat and other cereals [1][2][3]. A classical biological control program was implemented in North America against the invasive O. melanopus resulting in the deliberate introduction of the European fairyfly Anaphes (Anaphes) flavipes (Foerster) (Hymenoptera: Mymaridae), in natural conditions a known egg parasitoid of Lema Fabricius and Oulema species [4]. As the result, A. flavipes is now well established in Canada and the USA where it is considered to be an important biological control agent. Additionally, attempts of a neoclassical biological control were made by evaluating, under quarantine laboratory conditions in Washington State, USA, Anaphes nipponicus Kuwayama from Fujian, China [5,6], which readily attacked, oviposited and successfully completed two generations on eggs of O. melanopus; however, due to inability to develop successfully at low humidity it was not considered to be a promising biocontrol agent in eastern Washington [6]. Anaphes nipponicus was originally described from Hokkaido Island, Japan, as an egg parasitoid of the rice leaf beetle Oulema oryzae (Kuwayama) (Fig 1A), a pest of rice mainly in the temperate zones of East Asia, particularly in its Palaearctic parts. This parasitoid species was originally described from a syntype series from the following two type localities: Ō no in Kameda District (now Hokuto in Oshima Subprefecture of Hokkaido Prefecture), and Kagura, Kamikawa District (now Higashikagura in Kamikawa Subprefecture of Hokkaido Prefecture) [7]. The coordinates of these type localities, as indicated by Huber & Thuróczy (2018) [8], seem to be incorrect, particularly of the Ō no site which are not on land. Besides Fujian, China, which is within the Oriental part of the Palaearctic region, A. nipponicus was also reared from eggs of O. oryzae in Primorskiy Kray of Russia [9][10][11]. Ironically, until recently the taxonomic identities of both A. flavipes and A. nipponicus were unclear, and the former nominal species in particular was often misidentified unless being reared from eggs of Lema or Oulema hosts on cereals [1,2]. For A. flavipes at least, that was mitigated by Samková et al. (2017) [2] when the species was thoroughly redescribed and illustrated, so it became more or less recognizable, considering difficulties of identifying Anaphes Haliday species in general. It was very important that the redescription included a thorough morphometric analysis indicating wide ranges for the key diagnostic features. At the same time, even though A. nipponicus was also partially redescribed and illustrated in Samková et al. (2017) [2] and Triapitsyn (2021) [12], the lack of well-prepared, freshly collected, reared material from the known host of this species, recognizing it remained a taxonomic problem because morphologically they are very similar. Although Samková et al. (2017) [2] indicated some very minor differences between these two nominal species of Anaphes, Triapitsyn (2021) [12] found them to be either within or very close to the known morphological variability ranges of A. flavipes, so in his key to the Palearctic species of the nominate subgenus of Anaphes, to which they belong, they were separated only by their geographical distribution and the host associations. It was also indicated that a genetic comparison of these two nominal species would be very desirable to confirm their possible conspecificity [12]. Without supporting molecular data, it was impossible to make a well-justified decision regarding their taxonomic status because of these notable differences in the distributional ranges, habitats and host associations, even though they attacked eggs of the same leaf beetle genus (Oulema). Here, we present and analyze such genetic data for both nominal species based on reared specimens that reveal their conspecificity.

Collection of samples
In Germany, specimens of A. flavipes were reared from eggs of Oulema sp. on barley in Aachen and its environs, North Rhine-Westphalia, in the general area of both the original type locality (es) of this parasitoid and also at and near the type locality of its neotype designated by Samková et al. (2017) [2]. Because we used the ethanol-preserved (stored in a freezer since June 2011) voucher specimens of their study, including a few from the same reared series as the neotype, the collecting methods described in Samková et al. (2017) [2] fully apply to our specimens from Germany as well and thus do not need to be repeated.

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In Japan, rearing of A. nipponicus from eggs of Oulema oryzae in paddy fields was conducted in two different locations on Honshu Island. In Shizuoka Prefecture, egg masses of O. oryzae were collected 2.June.2021 by R. Nakano on rice plants in 4 non-organic paddy fields located within a 250 m radius (Table 1; Fig 1B) in Oyama, Sunto District. Each of the approximately 5 cm long pieces of rice leaves with O. oryzae eggs was placed in a 15 ml or 50 ml plastic centrifuge tube together with a crumpled piece of Kimwipes1 tissue (Fig 1C), and placed in an incubator at 20˚C, 50% relative humidity, and constant light from 7:00 to 23:00 in the laboratory (Fig 2A). The samples were checked daily for parasitoid emergence. The reared adult A. nipponicus were collected by T. Adachi-Hagimori from a few parasitized eggs of O. oryzae

Taxonomic studies
For morphological terminology we follow that of Triapitsyn (2021) [12]. An abbreviation for a funicular segment of female antenna is F. Molecular voucher specimens of A. flavipes were slide-mounted in Canada balsam using a slightly modified technique described in Huber (2015) [13]. All slide mounts were examined under a Zeiss Axioskop 2 plus compound microscope (Carl Zeiss Microscopy, LLC, Thornwood, New York, USA).
The following acronyms are used to designate depositories of voucher and other specimens examined: ELKU Entomological Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
UCRC Entomology Research Museum, University of California, Riverside, California, USA.

DNA extraction, amplification, sequencing, and genetic data analysis
Genomic DNA was extracted from nine individual wasps (4 A. flavipes from Germany and 5 A. nipponicus from Japan) using the non-destructive HotSHOT method of Truett et al. (2000) [14] in a total volume of 50 μL. Following DNA extraction, all specimens were retrieved and individually slide-mounted for morphological examination (see above). Each molecular voucher specimen was assigned a P. F. Rugman-Jones' molecular voucher PR number and UCRC database unique identifier number. Extracted DNA was stored at -20˚C. Three separate loci were amplified using the polymerase chain reaction: the 5' region of mitochondrial cytochrome c oxidase subunit I (COI) using the LCO1490 and HCO2198 primer set as described in Triapitsyn et al. (2021) [15]; and, the internal transcribed spacer 2 (ITS2) and D2 region of

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EMBOSS Transeq [18] was used to translate the protein coding COI sequences into their amino acid chains, confirming the absence of indels and pseudogenes in the final dataset. Short flanking sequences of 5.8S and 28S were identified, and subsequently removed, using the "annotate" function in the online ITS2 database [19,20]. Given the limited aims of our study, genetic analyses were restricted to simple comparison of DNA sequences of each locus among the sequenced specimens, and with those held in two public repositories, GenBank [21] and

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BOLD [22]. Sequences of each locus were aligned in MAFFT online using the Q-INS-I strategy. All sequences generated herein were deposited in GenBank.

Sequence analysis
We obtained 28S-D2 and ITS2 sequences from all 9 specimens, and a COI sequences from 8 of the 9 specimens. The sequences generated from the 4 individuals of A. flavipes from Germany were found to be almost identical to those of 5 individuals of A. nipponicus from Japan. Sequences of 28S-D2 were identical in length (520 bp) and nucleotide composition across all 9 specimens (GenBank accessions OM701783-701791) with the exception that a single German specimen (PR21-489) was polymorphic at a single position (S1 Table). Sequences of the typically much more variable ITS2 were also very similar in both length (514-517 bp) and composition with variation limited to a handful of single base substitutions or the insertion/deletion of a microsatellite repeat motif (OM701774-701782; S2 Table). No significant matches (>97%) were found in GenBank for 28S-D2 or ITS2. Sequences of the COI of the five specimens of A. nipponicus were identical (OM687258-687262) and these differed from those of three A. flavipes (OM687255-687257) specimens at only 3 positions (each a synonymous substitution; positions 148, 319, and 337; S3 Table). One of those positions (319) was also variable within A.
flavipes, but at a maximum of <0.5%; variation among the sequences was well within that expected at the intraspecific level. Taken together, sequences of these three loci provide conclusive evidence that the specimens of A. flavipes and A. nipponicus represent a single species. Comparison of the COI sequences with BOLD revealed the existence of several "private" accessions (identified only to genus) originating from specimens apparently collected as far and wide as Canada, Germany, Bangladesh, and Vietnam.    [2] based on specimens from Germany, from which a lectotype was designated. Triapitsyn (2021) [12] provided diagnoses and illustrations of both A. flavipes (based on specimens from Europe) and A. nipponicus (based on specimens from Wajima, Ishikawa Prefecture, Japan). Here we provide illustrations of the habitus (Figs 3A, 3B and 4A), antenna (Fig 4B and  4C), fore and hind wings (Fig 5A), metatarsus (Fig 5C and 5D) and ovipositor (Fig 5B and 5C) of the newly collected, good quality reared specimens from Japan to facilitate its recognition. In these slide-mounted specimens, body length 0.6-0.72 mm; antenna (Fig 4B and 4C) with F2 length very variable (sometimes very short in smaller specimens, Fig 4C), 2.4-4.0× as long as wide, and the combined length of F1 and F2 from slightly shorter than F3 to about as long as or slightly longer than F3, funicle with multiporous plate sensilla only on F3-F6 (2 on each), clava 3.5-3.6× as long as wide, a little shorter (0.9-0.95×) than the combined length of F5 and F6, with 6 multiporous plate sensilla; fore wing ( Fig 5A) 0.57-0.72 mm long, 6.9-7.0× as long as wide, longest marginal seta 1.3-1.4× maximum wing width, marginal space separated from medial space by 1 complete line of setae; metatarsomere 1 at most about as long as metatarsomere 2 (Fig 5C and 5D); ovipositor occupying 0.75-0.8× length of gaster (Fig 5B and 5C), not exserted beyond its apex, and about 1.1× as long as metatibia.
Various aspects of biology, ecology and parasitism of A. flavipes in Europe were studied by Donev (1987) [27] and Samková et al. (2019aSamková et al. ( , 2019bSamková et al. ( , 2019cSamková et al. ( , 2020Samková et al. ( , 2021 [3,[28][29][30][31]. REMARKS. Triapitsyn (2021) [12] looked for the missing syntypes of A. nipponicus in the collection of Insect Museum, National Institute for Agro-Environmental Sciences, NARO, Tsukuba, Ibaraki, Japan (ITLJ), to where S. Kuwayama's collection had been moved [2], during a brief visit in November 2019, but could not locate any. These appear to be lost. However, conspecificity of our specimens from Honshu Island with Kuwayama's A. nipponicus from the nearby Hokkaido Island is not in doubt as they were reared from eggs of the same beetle host in a similar habitat of a paddy field, and are identical to the other known specimens of this species [2,12,24].

Discussion
Morphologically, the newly reared specimens of A. nipponicus from Japan largely fit the known ranges of the diagnostically important features of both A. flavipes from Europe and A. nipponicus from Japan, as indicated in Samková et al. (2017) [2] and Triapitsyn (2021) [12]. Genetically, our analysis of the sequences of the selected mitochondrial and nuclear ribosomal gene regions unambiguously showed that these two nominal species are practically identical, well within an expected intraspecific genetic variability. Thus, their conspecificity is not in doubt, hence the synonymy of A. nipponicus under the much earlier described A. flavipes. Application of molecular methods for insect diagnostics has made it possible to resolve almost a century old misidentifications of these economically important natural enemies of key cereal agricultural crops.
In this particular case, identity of the A. flavipes individuals from Europe from which DNA was extracted, and then selected mitochondrial and nuclear ribosomal gene regions were sequenced, was not in doubt because these came from the same rearings and collections near Aachen, Germany, as the neotype (the ultimate identity defining specimen mounted on a slide) and other specimens on which morphological redescription of this species was based [2]. Therefore, this unfunded investigation was focused solely on revealing the true identity of A. nipponicus from Japan, which was not clear before this study, rather than on determining genetic variability of A. flavipes in Europe, that by itself warrants a separate study which would require substantial funding and effort to rear this egg parasitoid from different hosts throughout its range.
Although this conclusion about conspecificity of A. flavipes and A. nipponicus might be somewhat surprising considering their different geographical distribution, habitats and host associations (albeit parasitizing different species in the same host genus, Oulema), as well as obvious ecological differences of the respective agroecosystems between the cereal crops in Europe and paddy fields in temperate East Asia, similar examples do occur in Mymaridae. For instance, Anagrus incarnatus Haliday from Europe was found, using similar molecular methods, to be conspecific with A. nilaparvatae Pang & Wang from Asia, a well-known egg parasitoid of rice leafhoppers and planthoppers (Hemiptera: Cicadellidae and Delphacidae, respectively) [32].
Based on our collection data, parasitism of O. oryzae eggs on rice plants in the two sampled localities on Honshu Island in Japan seemed to be very low: for instance, out of the total 144 egg masses (individual eggs were not counted) of the leaf beetle host in the four paddy fields in Oyama, Shizuoka Prefecture, each of which contained several eggs, only one female and 2 male adult A. flavipes wasps emerged from one egg mass (Fig 2B) from the Oyama_3 field ( Fig  1B; Table 1). Thus, the actual parasitism rate of O. oryzae eggs by A. flavipes in these sampled rice fields was less than 1%. That, however, might be due to the fact that these were nonorganic, conventional rice fields with a history of prior pesticide use. Togashi (1974) [24], however, indicated 1.8-38.3% egg parasitism of O. oryzae in Ishikawa Prefecture, on the same island, mentioning that the highest percent parasitism at Tsurugi-machi (37.5%) and one area of Wajima City (38.3%) could have been due to preservation of the paddy fields in ancestral condition with more ecological diversity, thus probably providing a better habitat for the overwintering parasitoids.
Supporting information S1 Table.